The first year of the project involving the tissue factor pathway inhibitor (TFPI), previously referred to as LACI) established the factor Xa-inhibition assay and the TFPI purification method from hepatoma cells. Since that time the purification method has been applied to the production of sufficient quantities of material for immunization of rabbits to develop antisera and to be used in immunization of mice for the development of monoclonal antibodies. Initial fusion wells were assayed by both an ELISA assay as well as by an activity assay. Three hybridoma clones have been isolated, expanded and cloned and they have been demonstrated to produce IgM antibodies which block TFPI activity. These clones are being characterized for binding characteristics to the protein as well as their usefulness in an ELISA assay for TFPI. Rabbit antisera to TFPI was used in Western blot experiments to identify TFPI in crude preparations. In addition, the antisera was used to demonstrate the importance of TFPI in the dilute thromboplastin prothrombin time. In the standard prothrombin assay, both hemophilic plasma and normal plasma clot similarly. When the thromboplastin activator is used in lower concentrations, the hemophilic plasma exhibits a prolonged clotting time (CT). This CT can be decreased by the addition of rabbit ant-TFPI. We have shown that this is dependent on the amount of anti-TFPI added to the plasma. Intra-laboratory collaborative efforts with Dr. Fricke have involved the use of the TFPI in studies of its ability to block xa catalyzed activation of Factor VIII.